Source: E. coli MG1655 genomic DNA
Before starting design, you should read this tutorial:
A Guide to Circular Permution
In general, you should clone the bits and pieces out of genomic DNA first, subclone and sequence those bits,
then assemble them into full length in a separate step. You can use either SOEing as described in the tutorial to do
the assembly, or use standard assembly.
The reference for linker composition of eCPX is: PMID: 18480093
This part encodes a N-terminal display protein
Your displayer part should be of the {<part!} style (no start, with stop). There should be NO prepro sequence in your part.
You should design your construction file to insert your part into plasmid pBca9495KC-Bca1144#5 using EcoRI and BamHI.

- Ran an analytical gel to make sure pcr worked
- Did a zymo clean up on the PCR product to obtain the required fragments
- Digested the fragments with EcoRI and BamHI (according to the construction file)
- Cleaned up the digest
* Did not have time to finish ligating and transforming. So, Digests were stored in the freezer until the next lab period

*Set up the following reaction:
8uL of eluted PCR product
1uL of NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI
*Incubate at 37 degrees on the thermocycler for 1hr
*Run an agarose gel, and melt with 600uL ADB buffer at 55 degrees.
*If the DNA is shorter than 300bp, add 250uL of isopropanol and mix prior to loading it on the column

- Ligated the digests from the last lab period - Feb 23rd
- Transformed them into DH10B cells and put the plates in the incubator

LIGATION

*Set up the following reaction:
6.5uL ddH2O
1uL T4 DNA Ligase Buffer (small red or black-striped tubes)
1uL pBca9495AK-Bca1144
1uL Insert digest
0.5uL T4 DNA Ligase
*Pound upside down on the bench to mix
*Give it a quick spin to send it back to the bottom of the tube
*Incubate on the benchtop for 30min
*Put on ice and proceed to the transformation

TRANSFORMATION

Competent cells are stored as 200uL aliquots in the -80 freezer as a communal stock
1. Thaw a 200 uL aliquot of cells on ice
2. Add 50 uL of water
3. Add 30 uL of KCM salts
4. Put your ligation mixture on ice, let it cool a minute or two
5. Add 75 uL of the cell cocktail to the ligation, pipette up and down gently to mix
6. Let sit on ice for 10 min
7. Heat shock for 2 min at 42
8. Put back on ice for 1 min
9. For ampicillin selection, you can plate immediately, otherwise:
10. Add 100uL of LB, let shake in the 37 degree incubator for 40 min
11. Plate on selective antibiotics, let incubate overnight

- Colonies were picked and grown out for us over the weekend
- Today: Miniprepping!!!
- I had a total of 4 miniprep samples: 2 of each kind (The N and C termini)
- After miniprepping, a restriction mapping was performed to see if the samples should even be sent to the sequencing facility.
- Restriction mapping: Worked!
- Did not have time to send the samples for sequencing today - Will do them on the next lab period

MINIPREP PURIFICATION OF DNA

1. Pellet 1.5 mL saturated culture by spinning full speed, 30 seconds.
2. Dump supernatant, repeat to pellet another 1.5 mL (for a total of 3 mL)
3. Add 250uL of P1 buffer into each tube. Resuspend the cells using a vortexer.
4. Add 250uL of P2 buffer (a base that denatures everything and causes cells to lyse). Gently mix up and down. Solution should become
clearer.
5. Add 350uL of N3 buffer (an acid of pH ~5 that causes cell junk - including protein and chromosomal DNA - to precipitate, and
leaves plasmids and other small molecules in solution). Slowly invert a few times, then shake.
6. Spin in centrifuge at top speed for 5 minutes.
7. Label blue columns with an alcohol-resistant lab pen.
8. Pour liquid into columns, and place the columns into the centrifuge. Spin at 12000 rpm for 30 seconds.
9. Dump liquid out of the collectors under the columns (the DNA should be stuck to the white resin)
10. Wash each column with 500 uL of PB buffer.
11. Spin in centrifuge at 12000rpm for approximately 15 seconds, then flick out the liquid again.
12. Wash with 750uL of PE buffer (washes the salts off the resins).
13. Spin in centrifuge at 12000rpm for approximately 15 seconds and flick out liquid again.
14. Spin in centrifuge at full speed for 1 minute to dry off all water and ethanol.
15. Label new tubes and put columns in them.
16. Elute them by squirting 50uL of water down the middle of the column (don't let it stick to the sides).
17. Spin in centrifuge at top speed for 30 seconds.
18. Take out columns and cap the tubes.
19. Clean up - note the P1 buffer is stored at 4degC and all the rest at room temperature.

RESTRICTION MAPPING

3uL of miniprep DNA
5ul H20
1uL of NEB Buffer 2
0.5uL EcoRI
0.5uL BamHI
*Incubate at 37 degrees on the thermocycler for 1hr
*run out on a gel to check size (should be 2974bp, 282bp)

- Samples were sent in for sequencing
- However, to avoid wasting time, the 3rd part of the construction file - SOE-ing PCR was started today
- N and C termini constructs were not verified to be correct
- But only the PCR was set up such that if the first two constructs were wrong, only the SOE-ing PCR step would be a waste

- Since the first two constructs were verified to be correct, I proceeded with SOE-ing PCR
- Performed a zymo clean up of the PCR
- Did a restriction digest
- Ran the samples on a gel and gel purified the required fragments (cut out the bands with a razor before purification)
- Once fragments 'A' and 'B' were obtained (check construction file), a second PCR was set up with the SOE-ing oligos on A+B

- Today was a very short day:
- I picked a colony from the plates I had transformed and put it into the Shaker
- (Gabe said he would monitor the test tubes + miniprep my samples over the weekend)
- Plan - To send the samples in for sequencing first thing on Monday

Plan:
- To transform RFP into DH10B cells
- These cels would be plated on Spec plates'
- Colonies would be picked from these plates and cultures grown - they would be co-transformed with the IILK and AG4 plasmids
- We will also make clones without the RFP plasmids

- Performed 1:10 Dilutions
- 96-well plate: 1 mL of LB (with or without arabinose) and 100uL of saturated cell culture.
- In the V-bottom plate: 300uL of LB (with or without arabinose) and 30uL of saturated cell culture.
- The V-bottom plate would allow us to qualitatively assess whether any flocculation occurred.
- The 96-well plate was used because RFP measurements could easily be made using the Tecan.

- Due to inconclusive results, certain parts were re-transformed.
- The idea of co-transforming with RFP was not used.
- Instead, it was decided that sodium hydroxide could be used to get rid of cell clumps from flocculation
- Breaking up the clumps was necessary to obtain an accurate concentration of cells.